• Journal of Applied Biological Chemistry
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• v.63 no.4
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• pp.291-295
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• 2020

In order to increase the utilization of defatted sesame meal (DSM), a by-product of sesame oil production, the conditions of extraction of insoluble proteins from DSM by enzyme treatment were investigated. As a result of comparing the treatment results of proteolytic enzymes Alcalase, Flavorzyme, Neutrase, and Protamex with control, Protamex was effective in increasing the total solid and protein content. At the reaction conditions of Protamex (50 ℃, pH 6.0), the dosage of enzymes was appropriate for 1% of DSM and 3 h of enzyme reaction time. To improve the efficiency of enzymatic treatment, the protein content extracted increased as the heat treatment temperature increased, and slightly increased above 110 ℃. As a result of investigating the effect of the combination treatment of cell lytic enzyme (Tunicase) and protease (Protamex) on protein solubilization, it was most effective to treat the cell lytic enzyme after processing the protease. After heat treatment (110 ℃, 10 min), sequential treatment of Protamex and Tunicase increased the protein content by about 3.5 times (9.85→35.58 mg/mL) of the non-heated control and 2.2 times (15.83→35.58 mg/mL) of the heat treated control.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.3
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• pp.230-237
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• 2018

This study investigated the food and nutritional characteristics of alkaline-insoluble fractions (AIFs) recovered from bastard halibut Paralichthys olivaceus (BH) and skipjack tuna Katsuwonus pelamis (ST) roes using the alkaline solubilization. The moisture content of AIFs ranged from 4.8% to 12.8%, and ST provided significantly better yields (9.5 for STAIF-11 and 7.1 g/100 g roe for STAIF-12) than did BH (P<0.05). The protein content of AIFs ranged from 71.7% to 79.2%, with the highest level yielded by STAIF-11 (6.8 g/100 g roe). The crude fat content of AIFs was 10.9-14.3% and the mineral content was 0.7-3.4%. The major mineral components of AIFs were sulfur, sodium, potassium, and phosphorus. Color values showed that BHAIFs were significantly brighter than STAIFs. Total contents of essential amino acids were significantly higher in STAIFs (47.5-49.5%) than in BHAIFs. The major essential amino acids found in AIFs from both sources were Val, Leu, Lys, and Arg. Therefore, AIFs were significantly superior to whole BH roe in terms of physicochemical and nutritional status, and we identified species-specific differences between BH and ST. Protein is a major component of AIFs recovered from fish roes, which suggests that they have potential for use as a protein source.

• Korean Journal of Food Science and Technology
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• v.26 no.5
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• pp.490-495
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• 1994

Enzymatic solubilization of tofu-residue was attempted using carbohydrase isolated from Aspergillus niger CF-34. Tofu-residue, by-product of tofu manufacture or soymilk processing was used as the model for plant cell wall. It was found that tofu-residue was rich in nurients: 46.7% carbohydrate, 32.8% protein, the rest being lipid and ashes. Carbohydrate component of tofu-residue consisted of 36.8% cellulose and 62.6% hemicellulose. The carbohydrase was found to consist of pectinase, xylanase, PGase, CMCase, and SFase when tofu-residue and pectin were used as the carbon source. Enzyme induction was maximum at 7days of culture. Optimum reaction pH was 4.0, temperature $50^{\circ}C$. The enzyme was stable to $50^{\circ}C$, above which the stability decreased rapidly.

• KSBB Journal
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• v.14 no.4
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• pp.429-433
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• 1999

$\beta$-Glucosidaes from Cellvibrio gilvus(CG) was successfully overproduced in soluble form in E. coli with the coexpression of GroEL/ES/. Without the GroEL/ES protein, the $\beta$-glucosidase overexpressed in E. coli constituted a huge amount(80%) of total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the $\beta$-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. Similar effects of GroEL/ES were also observed on the overexpressed $\beta$-glucosidase from Agrobacterium tumefaciens(AT). And pET28(a)-RGRAR, partially deleted mutant lacking 5-amino acid residues at carboxy teminus also could be folded into an active form when expressed with the molecular chaperonin GroEL/ES, and its activity was higher than that of the without GroEL/ES system, In addition, the synergistic effect of GroEL/ES and the low induction temperature were important factors for solubilization of the inclusion body from overproduced $\beta$-glucosidases.

• Korean Journal of Food Science and Technology
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• v.23 no.6
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• pp.695-701
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• 1991

Spent layer breast meat and leg meat samples washed with 0.05 M sodium bicarbonate solution and 0.04 M phosphate buffer(pH 8.3) showed decreases in heat denaturation temperature indicating the destabilization of myofibrillar proteins. The destabilization was attributed to the solubilization of 95 Kdalton and 55 kdalton proteins from the myofibrils observed in gel-electrophoretograms. Transmission electron microscopy further indicated the breakage of Z-lines.

• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.137-143
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• 1997

Porcine transforming growth factor-$\beta$1 (TGF-$\beta$1) was expressed in Escherichia coli using cDNA of TGF-$\beta$1 and glutathione S-transferase (GST) fusion vector pGEX-1$\lambda$T. An ApoI-Tth111I fragment of cDNA which correspond to the amino acid residues from 123 to 390 of the precursor TGF-$\beta$1 was inserted into EcoRI-Tth111I digested pGEM#-l$\lambda$T and the recombined plasmid was named pGET-12. Gene products from the cloned regions of the recombinant plasmids pGET-12 was not detected in soluble fraction of cell free extract but detected in insoluble fraction. The solubilization of insoluble gene product was achieved by the treatment of N-laurylsarcosine. Molecular weight of partially purified proteins determined by electrophoresis was same as expected from cloned fragment. The ELISA test results of the purified proteins showed that immunologically detectable epitope was preserved in recombinant protein.

• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.559-562
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• 1998

Odontoglossum ringspot virus (ORSV) was finally purified from ORSV-infected orchid plants by diethylaminoethyl (DEAE) cellulose anion exchange column chromatography. The virus was reliably eluted by potassium chloride at the concentration from 0.1 M to 0.13 M. Partial purification was done by solubilization with Triton X-100 (allkylphenoxypolyethoxy ethanol) and precipitation with polyethylene glycol (PEG; MW 8,000). The finally purified ORSV represented one distinct homogeneous band and the molecular weight of its capsid protein was about 17,500 Dalton in electrophoretic analysis. Electron microscopy showed not only intact particles ranged from 280 nm to 340 nm in length, but also segmented particles that final 140 nm to 220 nm and even disks. Enzyme-linked immunosorbent assay (ELISA) showed that final yield was 12 mg/100 g of the infected leaves. Bioassay demonstrated that the purified ORSV had the normal infectivity to orchid plants and Nicotiana glutionsa. Based on these data, anion exchange column chromatography could be efficiently applied to the purification of ORSV and other viruses similar to ORSV.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.56 no.1
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• pp.21-27
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• 2023

Lotus Nelumbo nucifera seed protein (LSP) was isolated by alkaline solubilization after removing fat and phenolics by hexane and ethanol treatment. Antioxidant peptides from LSP were produced with Alcalase^® and pepsin and hydroxyl radical scavenging activities were determined. LSP-Alcalase^® hydrolysates showed higher hydroxyl radical scavenging activity than LSP-pepsin hydrolysates. To purify antioxidant peptides, LSP-Alcalase^® hydrolysates were subjected to high performance liquid chromatography (HPLC) separation on the C18 column and the active fraction was further purified using a Superdex^TM peptide 10/300 GL column. Finally, the active fraction (F8-2) was evaluated for antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging, and oxygen radical absorbance capacity (ORAC) assays. The EC₅₀ values of the F8-2 were 105.81±0.02 ㎍/mL for DPPH and 32.26±0.02 ㎍/mL for hydroxyl radical and the F8-2 exhibited 7.22 μM trolox equivalent (TE)/100 ㎍ F8-2. Glutathione (GSH), which is a positive control, showed EC₅₀ values of 19.87±0.01 ㎍/mL for DPPH and 15.95±0.03 ㎍/mL for hydroxyl radical and an ORAC value of 14.17±0.03 μM TE/100 ㎍ GSH. Finally, sixteen peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Ile-Tyr and Leu-Tyr showed higher antioxidant scores.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.700-704
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• 1998

Each of the recombinant structural genes of Helicobacter pylori urease, ureA and ureB, was cloned and overexpressed as inclusion bodies. Solubilization and renaturation of the inclusion bodies were carried out, to accelerate the pairing of sulfhydryl groups and the incorporation of nickel ions, which would lead to the native structure with high enzyme activity. Rates of urea hydrolysis were monitored as an indication of in vitro activation of renatured ureases. The activation of the apoprotein using 1 mM nickel ion, 100 mM sodium bicarbonate and a 10:1 ratio of reducing power resulted in a weak urease activity (about 11% of the native urease activity encoded by pTZ 19R/ure-l). When a sparse matrix screen method originally discovered for the crystallization of proteins was used, the activity increased higher than that obtained using glutathione. The effect of polyethylene glycol (PEG) on the activity was noticeable, giving two-fold increase in the specific activity (about 11 U/mg of protein corresponding to 22% of the native urease activity encoded by pTZ19R/ure-1).

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.216-219
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• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.